Design and Development of Reverse Slot Blot for the Simultaneous Detection of Rare and Regional Specific Mutations in the Beta Globin Gene in Khuzestan Province of Iran

Abstract

Beta-thalassemia is the most frequent hemoglobin disorder in Iran resulting from disrupting mutations in the beta globin (HBB) gene that causes decreased or complete absent of beta-globin chains. The screening of beta-thalassemia minor and major individuals and prenatal diagnosis is important for familial planning. Therefore, it is essential, depending on the ethnicity and local frequency of changes, to develop a rapid and accurate method for molecular diagnosis of beta-thalassemia. Here, we developed reverse slot blot (RSB) assay for the simultaneous detection of six common pathogenic changes in the HBB gene (-88, -28, IVSII-745, IVSII-848, Codon 6 [G → A] for HbC, Codon 6 [A → T] for HbS) in the Khuzestan Province of Iran. We designed normal and mutant oligonucleotide probes for each selected mutation and fixed them on positively charged nylon membrane. In the next step, a multiplex-polymerase chain reaction (PCR) performed for the amplification of the entire HBB gene using labelled 5′-biotinylated primers. The PCR products were hybridized to immobilized oligonucleotide probes on the membrane at the appropriate temperature. Finally, we developed the membrane by chemically colorimetric reaction using nitro-blue tetrazolium-5-Bromo-4-chloro-3-indolyl phosphate. For the best probe concentration, we made a serial dilution of probe pairs for each mutation. The optimal probe concentration for each mutation varied from 25 to 50 pmol. In the next step, DNA samples from homozygous affecting individuals were subjected for multiple PCR. Hybridization of each PCR products on the nylon membrane with probe pairs revealed specific bands with expected signal intensity without any background. Our designed RSB test is a rapid, sensitive and cost-effective method for screening of regional specific beta-thalassemia mutations in the Khuzestan population of Iran, which might be extended for the detection of any desired pathogenic changes.

Background

Beta thalassemia is a common hemolytic disorder in the Middle East with an autosomal recessive inheritance. This kind of anemia has higher prevalence in the Mediterranean region, South-East Asia, west and North Africa [1], compared to the Europe and the USA with lower frequency. It is also very common in north and southwest of Iran [2]. The prevalence of beta-thalassemia is about 10% around the Caspian Sea and the Persian Gulf, but with 4–8% much less in the other parts of country. Over two million beta-thalassemia carriers and almost 25,000 patients are estimated country-wide [3]. The disease is usually caused by more than 200 identified pathogenic changes within coding and non-coding parts of the HBB gene that leads to decreased or absent synthesis of the beta-globin chain [4]. Although there are almost 50 different mutations identified in the HBB gene in Iranian population, some are rare, depending on ethnic regions of the country. For instance, the mutation cd36–37 counts as the most common changes in an Arabian ethnic group in southwest Iran [4]. Therefore, it is necessary to evaluate specific methods for molecular diagnostic of beta-thalassemia in the laboratory [3, 5].

The best diagnosis method for screening of the beta-globin gene should be simple, rapid, cost-effective, and sensitive without need for advanced equipment. Presently, the important molecular diagnostic methods are used for the detection of the HBB mutations, based on polymerase chain reaction (PCR), such as reverse dot or slot blot [6], amplification refractory mutation system (ARMS) [7], polymerase chain reaction/restriction fragment length polymorphism analysis (PCR–RFLP) [8] and real-time PCR [9]. However, these methods are either expensive or time-consuming or may have false results.

A routine approach for simultaneous detection of beta-thalassemia causing mutations is reverse dot blot (RDB) or reverse slot blot (RSB) [8–10] as a main approach for diagnosis of many disease causing pathogenic mutations in human, such as cystic fibrosis, β-thalassemia, some cancer causing oncogenes as the EGFR and K-ras genes [10, 11]. The RDB test is based on fixation of oligonucleotide probes on the surface of nylon membrane. Usually, wild type (WT) and mutant (MT) oligonucleotide probes are immobilized on the solid surface. Then, the amplified sample from targeted gene region, labeled using 5′-biotin-primers or DIG-11-dUTP, hybridizes with blotted fixed probes on the surface. The RDB is the rapid, sensitive, nonradioactive and cost-effective method that might be ideal for screening of several pathogenic mutations, simultaneously [12, 13]. There is a high spectrum of beta-thalassemia mutations in Khuzestan province, in neighborhood of the Persian Gulf and Arabic country, due to high migration rate from different ethnic populations [14, 15]. We aimed also to design a test with above-mentioned properties for screening of beta-thalassemia patients in the Khuzestan Province.

Methods

Fifty-five DNA samples belonging to the beta-thalassemia majors with the patient’s consent have been collected from Shafa hospital in Ahvaz, Iran over a period of 6 months.

Samples had previously undergone molecular genetic diagnosis by sequencing, so that we knew their mutations in the HBB gene. In parallel to the patient’s samples, thirty samples from healthy people without any mutations in the HBB gene have been used to test the specificity and accuracy by homemade RSB. Results have been compared to commercial RDB strip.

Multiplex PCR Amplification

After genomic DNA extraction by salting out method, the HBB gene was amplified using multiplex PCR (M-PCR) by 5′-Biotin labeled Primer (Table 1). Polymerase chain reaction was performed by thermal cycler (Master-cycler Gradient, Eppendorf, Germany) in 25 μL reactions containing 100 ng genomic DNA, 1X PCR buffer, 0.2 mmol L⁻¹ dNTP, 1.5 mmol L⁻¹ MgCl₂, 2.5 units Smart Taq DNA polymerase (EuroClon company) and appropriate amount of primers (Table 1). The PCR condition consisted of an initial denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 15 s, annealing at 58 °C for 30 s and extension at 72 °C for 1 min 30 s; then, a final extension step at 72 °C for 5 min. Finally, the PCR products were analyzed by electrophoresis on 1.5% agarose gel.

Table 1.

List of primers used for the amplification of the entire HBB gene in this report

PCR primer	Concentration (pmol L−1)
CH01: 5′-Biotin-GTACGGCTGTCATCACTTAGACCTCA-3′	0.4
PC03: 5′-Biotin-ACACAACTGTGTTCACTAGC-3′	0.4
PC06: 5′-Biotin-TCATTCGTCTGTTTCCCATT-3′	0.4
CH03: 5′-Biotin-GTGTACACATATTGACCAAA-3′	0.2
CH04: 5′-Biotin-AGCACACAGACCAGCACGTT-3′	0.2

Sequences of primer were used from previous literature [11]

Preparation of Test Strip

Strip preparation was done according to modification on previously reported method by Zhang et al. [16]. The 5′ amino-modified oligonucleotide probes were synthesized by the Macrogen company (Table 2) [11]. Probe dilution was performed by 0.5 M sodium bicarbonate buffer pH 8.4. Positive charged nylon membrane (PALL, Biodyne C) was pre-activated with a freshly prepared 16% EDC (1-ethyl-3-dimethyl aminopropyl-carbodimide, Sigma) for 10 min, with subsequent membrane washing in PCR based water and air-drying. After spotting of 25 pmol oligonucleotide probe on the membrane in the slot blot instrument that we designed and produced for this project (Fig. 1).

Table 2.

List of oligonucleotide probes used in this work with corresponding sequences and appropriate used concentration for each probe pair

Probe name		Concentration (pmol L−1)
Normal sequence
-88	5′-NH2-GGAGCCACACCCTAG-3′	25
-28	5′-NH2-GGGCATAAAAGTCAGG-3′	25
Codon 6	5′-NH2-TGACTCCTGAGGAGAAGT-3′	25
IVS 2745	5′-NH2-CAATCCAGCTACCATTC-3′	25
IVS 2848	5′-NH2-GGAGCTGTGGGAGGA-3′	25
Mutant sequence
-88	5′-NH2-ACCCTAGGATGTGGCT-3′	100
-28	5′-NH2-CCCTGACTTCTATGCCC-3′	100
Codon 6 (HbS)	5′-NH2-CAGACTTCTCCACAGGA-3′	25
Codon 6 (HbC)	5′-NH2-CAGACTTCTCCTTAGGAG-3′	25
IVS 2745	5′-NH2-GAATGGTACCTGGATTG-3′	25
IVS 2848	5′-NH2-CTCCCAAAGCTCCTGG-3′	100

Fig. 1.

Slot blot manifold designed, produced, and used for this study. a Fluid collecting container with outlet. b Slot plates with 1 mm diameter; nylon. c manifold parts with fixed membrane are tightly screwed before use

The activated membrane was quenched with 0.1 M NaOH solution for 10 min, followed with immersing in hybridization buffer (2 X saline sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS)) at 45 °C for 15 min. We added fifty-microliter PCR products to hybridization buffer in a final volume of 200 μl, denatured at 95 °C for 10 min, and set immediately on ice for 5 min. The denatured DNA was added to the pre-hybridized strip and incubated at 45 °C for 90 min in a shaking water bath (Memmert, Germany), washed twice in 2 X SSC and 0.5% SDS at 45 °C for 30 min and incubated in a solution containing streptavidin–alkaline phosphate (AP) conjugate for 25 min at room temperature. Strips were washing two time in 2X SSC and 0.5% SDS for 5 min at room temperature, and subsequently incubated to nitro-blue tetrazolium-5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) in the dark for 30 min at room temperature.

Results

We used multiplex PCR with five primer pairs for the amplification of the entire HBB gene. The length of the products were 1361, 739, and 423 base pairs, respectively. Primers were biotin labelled at the 5-prime terminal. To get best results, we compared the effect of two Taq DNA polymerase from amplicon and EuroClon companies. Reliable amplicons were reached by SMART DNA polymerase (EuroClon company), as we showed in Fig. 1.

In the next step, aimed to find out the optimum concentration of the oligonucleotide probes affecting signal intensity for wild type and mutant genotype. Thus, we investigated a serial dilution of probe concentration over the range of 0.5–100 pmol per μL⁻¹. The signal intensity of probes after hybridization with complimentary sequences was measured by image analyzing software. The lowest concentration with higher signal intensity but without false signal was used for samples (Fig. 2).

Fig. 2.

Multiplex-PCR products for the HBB gene. Line 1and 2 represent PCR amplicons with appropriate sizes amplified by Taq polymerase from Amplicon Company, compared to the SMART Taq DNA polymerase showed in line 4 and 5. Line 3 and 6 represent negative control

The same procedure made for each probe pair (wild type and corresponding mutant), the sequence and the optimal concentration for each probe pair is given in Table 2.

The prepared test strips for screening of HBB gene mutations were presented in Fig. 3.

Fig. 3.

Serial dilution of a selected probe pair from 0.5 to 200 pmol specific for the detection HbS-wild type (WT) and HbS-mutant (M)

To validate our tests, we picked fifty-five DNA samples with known genotype for the HBB gene from beta thalassemia majors or sickle cell anemia patients. All patients were previously genotyped by sequencing of the entire HBB gene. Six frequent mutations in southwest Iran were selected, such as -88 (C > A), -28 (A > C) locating on the promoter of the HBB gene, and IVSII-745 (G > C) and IVSII-848 (A > C) locating on the splice site of intron two. The two other mutations were HbS and HbC, which are locating on codon six, causing hemoglobin S and hemoglobin C production. As we can see in Fig. 4, we detected all expected changes in the HBB gene without any background or false positive or negative results, validated by post sequencing of the HBB gene. Signal intensity is continuously satisfying for all probes and concentrations. Optimal concentration for used DNA for the hybridization was at least 120 ng of PCR product.

Fig. 4.

Final result of RSB. Line: (1) -28/-28; (2) IVS-II-745/IVS-II-745; (3) IVS-II-848/IVS-II-848; (4) HbC/N; (5) HbS/HbS; (6) N/N. Sodium bicarbonate buffer was used as negative control

Discussion

Beta thalassemia is one of the most prevalent hemolytic disorders in Iran [4]. Application of standard molecular diagnostic methods is essential for the prenatal diagnosis of thalassemia and genetic counseling [17]. Several molecular techniques have been developed for diagnosis of this kind of anemia, such as dot/slot blot, PCR–RFLP, ARMS, microarray, real-time PCR [6]. Among these methods, RSB/RDB is the main molecular diagnostic method for screening of beta-thalassemia, because of its several advantages, including simplicity, speed, sensitivity, and also simultaneous detection of several mutations [12]. A recent research developed a reverse dot blot method for the detection of frequent alpha and beta thalassemia in a Chinese population [18]. Researchers evaluated the rapid and nonradioactive RDB chip for screening some alpha-thalassemia point mutations, in Italy [19].

We aimed here to develop the reverse slot blot assay as rapid and nonradioactive method for simultaneous detection of common mutations in the Khuzestan population, but rare in other geographic locations in Iran or Middle East. These mutations are often screening by ARMS method as rapid test, but with false negative results [14]. At present, DNA sequencing is the best method for the detection of pathogenic changes, which requires expensive and time-consuming instruments and equipment, and not usable everywhere.

There are already commercial RSB kits for screening some thalassemia mutations. However, commercial RSB do not cover most regional specific pathogenic changes such as -88 and -28 within the HBB gene promoter. Pathogenic changes in the HBB gene promoter are observed with relative higher frequency in Khuzestan Province [15]. Otherwise, the multiplex PCR method used in present study contributes timesaving diagnostic process, in comparison to the existing kits, which need two or three PCR reaction for productions of target DNA. In order to improve signal intensity, we labelled the biotin agent on both forward and reverse primers used in the PCR. However, some work had used only one biotin-primer at the 5-terminal [18]. In addition, and for more signal improvement, we used positively charged nylon membrane covalently bond to the oligonucleotide probes.

Our designed probes create reproducible and higher sensitivity results in comparison to the T-tailed probes that bond usually to the surface of membrane via UV cross-linking [16]. We proved the results of RSB tests by DNA sequencing, which showed no difference.

We conclude the successful design and development of a diagnostic rapid test for pathogenic mutations in the HBB gene that occur more or less in southwest Iran. As our results showed, we could use and expand this simple, rapid and nonradioactive test for any other mutations reported for the Khuzestan Province, such as IVSII-1 and cd36-37 variations, accounting for more than 30 percent of total pathogenic mutations in the HBB gene [15]. The slot-blot manifold specifically designed and developed for this purpose has the screening capacity of more than twenty-eight variations on a single strip.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.